OpticalFlow3D - a tool for measuring amorphous motion in three-dimensional fluorescence microscopy images

Motion is an essential component of any living system. It is rich with information, but it is often challenging to quantitatively extract biologically informative results from the motion apparent in microscopy images. This challenge is exacerbated by the wide variety in biological movement, which often takes the form of difficult-to-segment amorphous structures undergoing complex motion. An image processing technique known as optical flow can capture motion at each pixel in an image, thus bypassing the need for object segmentation or prior definition of motion types. This makes it a powerful tool for quantitative assessment of biological systems from the protein to organism scale. However, despite its flexibility and strengths for analyzing fluorescence microscopy images, its adoption in the bioimaging community has been limited by the availability of easy-to-use tools and guidance in results interpretation. Here, we describe an optical flow tool, called OpticalFlow3D, that can be run in Python or MATLAB and is compatible with three-dimensional microscopy images. Using biological examples across length scales, we illustrate how OpticalFlow3D can enable new biological insight.